Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-induced CTSB-dependent cleavage of Sialin at K256/R257/I258 generates Sialin2, which localizes to mitochondria a, Immunoblot analysis of Sialin and Sialin2 protein normalized to HSP90 in cerebral cortex from Alzheimer’s disease (AD) mice, salivary glands from irradiation (IR)- damaged mice. Representative data of n = 6 independent experiments were shown. See full immunoblot images and quantitation in Extended Data Fig. 1a. b, Endogenous Sialin2 was immunoprecipitated (IP’ed) from HEK293T cells, with IgG as negative control. Representative data of n = 3 independent experiments were shown. c, Immunoblot analysis of Sialin and Sialin2 in HEK293T cells pre-treated for 6 h with DMSO (vehicle), 200 nM Baf-A1/40 μM CQ (lysosomal inhibitors), or 10 μM MG132 (proteasome inhibitor), followed by 4 h stimulation with 4 mM nitrate. Representative images of n = 3 independent experiments were shown. d, Protein-protein interaction network (GeneMANIA) predicted cathepsin B (CTSB) as a top candidate protease interacting with Sialin. e, Co-IP of Sialin and CTSB in HEK293T cells treated with 4 mM nitrate for 4 h. Representative data of n = 3 independent experiments were shown. f, g, Proximity ligation assay (PLA) of Sialin/Sialin2-CTSB in HeLa (f) or NRK (g) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. h, Immunoblot analysis of Sialin and Sialin2 in HEK293T (left panel), HeLa (middle panel) or NRK (right panel) cells pre-treated for 6 h with a vehicle control (DMSO) and 20 μM CTSB inhibitor CA-074, followed by 4 h stimulation with 4 mM nitrate. Representative images of n = 3 independent experiments were shown. i, j, HEK293T (left panel) or HeLa cells (right panel) introduced with HA/Flag-tagged full length (FL) Sialin or mutant full-length Sialin (KRI/AAA) were treated with nitrate (4 mM, 4 h), followed by analysis of HA, Flag, Sialin, and Sialin2. Representative images of n = 3 independent experiments were shown. I, Ile; K, Lys; R, Arg. k, Top enriched cellular component GO terms for unique Sialin2-related proteomes in HEK293T cells listed by the rank of P values based on DAVID analysis. l, PLA of Tomm20-LAMP1 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. m, High-sensitivity structured illumination microscopy (HiS-SIM) live-cell images of Sialin or Sialin2 colocalized with MitoTracker-labeled mitochondria (PK Mito) in HeLa cells stably expressed GFP-Sialin or GFP-Sialin2. See quantification in Extended Data Fig. 2f. n, Immunoelectron microscopy images of Sialin or Sialin2 localized to mitochondria in NRK cells stably expressed GFP-Sialin and mCherry-Sialin2. N = 30 cells from representative experiments of three repeats. o, Schematic illustration of nitrate-activated CTSB cleaving Sialin at K256/R257/I258 to generate Sialin2, which is preferentially localized to mitochondria. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Mito-ABKAR (organelle-targeting sequence: MAIQLRSLFP LALPGMLALLGWWWFFSRKKA, N-terminus; Addgene: #61509) and Lyso-ExRai- AMPKAR (organelle-targeting sequence: full length of lysosome-associated membrane protein 1 (LAMP1), N-terminus; Addgene: # 192449) were created by Jin Zhang lab.

Techniques: Western Blot, Irradiation, Quantitation Assay, Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Control, Mutagenesis, Microscopy, Labeling, Stable Transfection, Immuno-Electron Microscopy

Journal: Andrology

Article Title: Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

doi: 10.1111/andr.13537

Figure Lengend Snippet: FIGURE 3 Characterization of C18-4Cox1-KD1 cells and KD-associated morphological and gene changes. (A) Expression of cyclooxygenase (Cox)1 on populations of transfected cells with varying concentrations of shRNA and lipofectamine 3000 showing that knockdown “B” had the most downregulated expression of Cox1. (B) Cox1 protein expression on immunoblot, comparing wildtype cells (WT), scrambled cells (Scrb), knockdown “B” clone, and an isolated clonal population of C18-4Cox1-KD1. C18-4Cox1-KD1 Cox1 protein expression validated against the total protein. (C) Gene expression of Cox1 in C18-4Cox1-KD1 and Scrambled controls, (D) Immunofluorescence staining of Cox1 in C18-4Cox1-KD1 and Scrambled controls. Green: Cox1, red: alpha-tubulin, blue: DAPI. Scale in µm. (E) Brightfield visualization illustrating morphological differences between C18-4Cox1-KD1 and Scrambled controls. (F) Differences in gene expression between C18-4Cox1-KD1 and Scrambled controls of Jam-1, Mmp2, Stra8, and Kit. Results are presented as fold change of controls. mRNA expression data were normalized to Glyceraldehyde 3-phosphate dehydrogenase (Gapdh); N = 3 independent experiments conducted in triplicates. Significant difference relative to controls with t-test: * (p ≤0.05), ** (p < 0.01), *** (p < 0.001).

Article Snippet: Cox1 shRNA plasmid for a mouse (sc-35097-SH) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotech.

Techniques: Expressing, Transfection, shRNA, Knockdown, Western Blot, Isolation, Gene Expression, Immunofluorescence, Staining

Journal: Andrology

Article Title: Differential roles of cyclooxygenase enzymes in the regulation of murine juvenile undifferentiated spermatogonia.

doi: 10.1111/andr.13537

Figure Lengend Snippet: FIGURE 5 Activation of Notch3 signaling pathway in C18-4Cox1-KD1. (A) Immunofluorescence (IF) images of Notch3 expression in C18-4Cox1-KD1 and Scrambled controls. Green: Notch3, red: alpha-tubulin, blue: DAPI. Scale in µm. (B) Immunoblots of Notch3, Hes1, cyclooxygenase (Cox)1, and Cox2 expression in C18-4Cox1-KD1 and Scrambled controls are cropped at indicated bands, and protein expression, indicated by values under the bands, is normalized to alpha-tubulin. (C) Quantification of Notch3, Hes1, Cox1, and Cox2 protein expression on western blot normalized against the total protein. Results are presented as fold change of controls. (D) IF images of Hes1 expression in C18-4Cox1-KD1 and Scrambled controls. Green: Hes1, red: alpha-tubulin, blue: DAPI. Hes1-positive cells were counted and the data show the percentage of Hes-positive cells as fold of control samples. N = 3 independent experiments. Significant difference relative to controls with t-test: * (p ≤0.05), ** (p < 0.01), *** (p < 0.001).

Article Snippet: Cox1 shRNA plasmid for a mouse (sc-35097-SH) and control shRNA plasmid (sc-108060) were purchased from Santa Cruz Biotech.

Techniques: Activation Assay, Immunofluorescence, Expressing, Western Blot, Control